Current models of horizontal gene transfer (HGT) in mycobacteria are based on "distributive conjugal transfer" (DCT), an HGT type described in the fast-growing, saprophytic model organism , which creates genome mosaicism in resulting strains and depends on an ESX-1 type VII secretion system. In contrast, only few data on interstrain DNA transfer are available for tuberculosis-causing mycobacteria, for which chromosomal DNA transfer between two strains was reported, a process which, however, was not observed for strains. Here, we have studied a wide range of human- and animal-adapted members of the complex (MTBC) using an optimized filter-based mating assay together with three selected strains of that acted as DNA recipients. Unlike in previous approaches, we obtained a high yield of thousands of recombinants containing transferred chromosomal DNA fragments from various MTBC donor strains, as confirmed by whole-genome sequence analysis of 38 randomly selected clones. While the genome organizations of the obtained recombinants showed mosaicisms of donor DNA fragments randomly integrated into a recipient genome backbone, reminiscent of those described as being the result of ESX-1-mediated DCT in , we observed similar transfer efficiencies when ESX-1-deficient donor and/or recipient mutants were used, arguing that in tubercle bacilli, HGT is an ESX-1-independent process. These findings provide new insights into the genetic events driving the pathoevolution of and radically change our perception of HGT in mycobacteria, particularly for those species that show recombinogenic population structures despite the natural absence of ESX-1 secretion systems. Data on the bacterial sex-mediated impact on mycobacterial evolution are limited. Hence, our results presented here are of importance as they clearly demonstrate the capacity of a wide range of human- and animal-adapted complex (MTBC) strains to transfer chromosomal DNA to selected strains of Most interestingly, we found that interstrain DNA transfer among tubercle bacilli was not dependent on a functional ESX-1 type VII secretion system, as ESX-1 deletion mutants of potential donor and/or recipient strains yielded numbers of recombinants similar to those of their respective parental strains. These results argue that HGT in tubercle bacilli is organized in a way different from that of the most widely studied model, a finding that is also relevant beyond tubercle bacilli, given that many mycobacteria, like, for example, or , are naturally devoid of an ESX-1 secretion system but show recombinogenic, mosaic-like genomic population structures.